[Au25(SR)18]- nanoclusters have been tested as a model catalyst in multiple oxidation reactions involving CO, alkenes, cyclohexane, and alcohols. Oxygen is used as an oxidizing agent in most of the reactions. Hence, O2 activation is of great interest in catalysis. The mechanism of these reactions, the role of intact nanoclusters as catalytically active species, and the utility of such nanoclusters as homogeneous catalysts are not completely clear. Herein, we investigate the interaction of [Au25(SR)18]- in solution with O2 using electrospray ionization mass spectrometry and density functional theory (DFT) calculations. Up to three O2 molecules attach to an [Au25(SR)18]- in dichloromethane (DCM) when O2 gas is passed through a solution of the former. Oxygen addition to the nanocluster leads to its decomposition. The nanocluster is most stable in toluene and least stable in tetrahydrofuran when kept under a continuous flow of O2, where no O2 adduct peaks are observed. It shows intermediate stability in DCM in the presence of O2, and the decomposition products, in this case, are of a different type compared to the former solvents. The appearance of O2 adducts and the variation in the stability of the nanocluster in different solvents is assumed to be due to the difference in oxygen solubility in these solvents. DFT calculations suggest that the first two O2 molecules interact with the surface Au atoms through the cavities formed by the ligands and staples in the nanocluster and the third O2 interacts only with 2-phenylethane thiol ligands. © 2018 American Chemical Society.